Corneal scarring and neovascularization (NV) following injury, trauma or infection affect 1.5 million Americans each year and are the 3rd leading cause of global blindness. Our central hypothesis is that decorin is the key regulator of corneal stromal wound healing, and that tissue-targeted localized delivery of therapeutic genes treats corneal disorders without significant side effects. Our long-term goal continues to be the development of tissue-targeted gene therapy approaches for corneal diseases in humans. We have recently developed targeted in vivo corneal gene therapy approaches using the adeno-associated vector (AAV) and simple topical delivery techniques. We found that localized AAV-decorin (AAV-dcn) therapy inhibits corneal scarring in vivo in rabbits without acute toxicity. Aim-1 of this proposal will test the hypotheses that targeted AAV-dcn therapy of rabbit keratocytes in vivo (a) inhibits their migration and proliferation by internalizing/ degrading epidermal growth factor receptor (EGFR) via caveolae-mediated endocytosis, and (b) limits myofibroblast formation by blocking transforming growth factor- (TGF) signaling without interfering with stromal collagen fibrillogenesis, which is critical for transparency. Further, we found that AAV-dcn decreases angiogenic vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1, and NV in rabbit corneas in vivo. The literature shows that VEGF-mediated angiogenesis can be counteracted by pigment epithelium derived factor (PEDF). This prompted us to hypothesize that combined decorin and PEDF delivery into rabbit keratocytes by AAV limits excess stromal healing, restores the cornea's normal physiological balance between pro- and anti-angiogenic factors, and eliminates corneal NV and concurrent scarring in vivo without adverse effects. Aim-2 will test this hypothesis by evaluating the efficacy and safety of localized topical combination (AAV-dcn+AAV-PEDF) therapy using an established rabbit alkali-burn induced in vivo corneal NV and scarring pathological model. Aim-3 will characterize molecular mechanisms by testing the hypothesis that AAV-dcn+AAV-PEDF therapy utilizes a dual mechanism wherein (1) PEDF eliminates corneal NV via Fas/Fas-ligand apoptosis; and (2) decorin inhibits NV by binding VEGFR2 and reducing VEGF-mediated ERK1/2 signaling, and further eliminates corneal scarring by blocking EGFR and TGF. We will accomplish these aims using human corneal fibroblasts, decorin knockout mice, a rabbit photorefractive keratectomy-induced corneal fibrosis model, an alkaline burn induced corneal NV and scarring pathological model, topical AAV gene transfer, immunofluorescence, immunoblotting, qPCR, apoptosis TUNEL assay, morphometric, and slit-lamp biomicroscopy. Successful completion of the proposed research will significantly advance the development of gene-based treatments for corneal NV and scarring, identify cornea-specific key fundamental mechanisms, and fill in gaps in our knowledge. Furthermore, our gene therapy approaches have a potential application in curing diseases in other tissues as AAV-dcn significantly inhibited peritoneum fibrosis in our collaborative studies with other scientists.